The ability to reprogram human fibroblasts and other cell types to full embryonic stem cell potential by transfer and expression of 4 genes in the recipient cells is a major technical advance for stem cell research and development. The reprogrammed iPS cells can self renew and differentiate to all cell types like human embryonic stem (hES) cells but because they are derived from adult skin cells they can be made from patient specific cells and thus offer the potential for genetically matched cell replacement therapies and drug toxicity testing. Moreover, patient specific iPS cell derivation will allow human cellular developmental models of many diseases to be made for the first time. With the number of pluripotent stem cell lines now increasing much more rapidly, it is even more urgent to develop efficient directed differentiation protocols. An important bottleneck is our current limited knowledge of surface markers that define the various cell types that differentiate from pluripotent stem cells. As pluripotent stem cells differentiate there is heterogeneity both spatially, in the cell types formed and temporally, in their degree of progression along their fated cell lineage. Even when cell culture conditions are manipulated to direct differentiation toward specific lineages there are still unwanted potentially tumor forming immature cells and cells of other lineages in the culture. The ability to define cell lineage and stage of differentiation by surface marker identification and cell sorting is needed to develop ways to isolate more homogeneous populations of iPS and other pluripotent cells for disease modeling, drug testing and discovery, and ultimately for preparing safe and effective cell therapies. It will also provide better understand of human development. We propose here to address this need by identifying new surface markers on iPS, and for comparison hES, derived cells and developing corresponding antibodies for cell identification and positive and negative cell selection. We will select surface protein encoding cDNAs from differentiating hiPS and hES cDNA libraries, using a cell survival based selection strategy by fusion to a drug resistance gene. The cDNAs will provide recombinant antigen and DNA expression plasmids for immunization. Antibodies will be raised in chickens because they are more likely to react to highly conserved antigens than mammalian hosts. The resulting antibodies will be tested on differentiated iPS and hES cells for their temporal and special expression and lineage specificity. Phase 1 will be a feasibility study to develop the techniques and prepare up to 15 antibodies. Phase 2 will expand the antibody development and identify additional surface antigens from lineage and stage specific cDNA libraries and disease specific iPS cells. The antibodies will be commercialized for use by the stem cell research and development community for defining pluripotent cell derivatives for disease modeling, drug testing and preparing cells for preclinical and clinical studies. . PUBLIC HEALTH RELEVANCE: Human embryonic stem (hES) cells have the potential to profoundly benefit public health by providing cells for treating virtually any degenerative disease or injury and as a source of human cells for drug testing and discovery. Embryonic stem cell-like cells have recently been derived from adult human skin cells using viral gene delivery. These induced pluripotent (iPS) cells are a potentially unlimited source of human cells of all types that are genetically matched to patients and can be used drug discovery, disease modeling and developmental studies, and ultimately cell therapies if they can be made safely without currently used viral vectors. Current methods of differentiating pluripotent cells result in mixed populations that can contain potentially tumor forming cells. Reagents and methods of defining specific subpopulations of induced and embryonic stem cell derived cells are needed. We propose here to develop antibodies to novel surface antigens on iPS and hES derived cells as reagents that will facilitate stem cell research and enable the isolation, scale up and production of cells for drug and cell therapy development.